The determination of enzymatically degradable substances; such as creatinine, glucose etc., by means of sensors in biological liquids, for example in blood, urine, plasma, serum and liquor, is preferably carried out via biosensors comprising immobilized enzymes. From the literature, several electrochemical and photometric methods of determining those substances are known.
In that way, creatinine may be potentiometrically determined, for example by means of the enzyme creatinine deiminase, involving a subsequent determination of the ammonium content. Another method consists in determining the creatinine concentration by means of an enzyme cascade using the enzymes creatininase, creatinase and sarcosine oxidase, with hydrogen peroxide (H2O2) being finally measured at an amperometric electrode (Tsuchida, T., Yoda, K., Clin. Chem. 29/1, 51-55 (1983)).
The invention relates to a method for calibrating biosensors that function in accordance with the last-mentioned principle. The conversion of creatinine to hydrogen peroxide is carried out according to the following reaction steps:
a.creatinine + H2O—creatininase→creatineb.creatine + H2O—creatinase→sarcosine + ureac.sarcosine + O2 +—sarcosine oxidase→glycin +H2O + H2O2formaldehyde
In order to be able to faultlessly determine the creatinine in a biological sample, it is necessary to correct the results of the creatinine system that consists of an electrode system comprising all three enzymes by those of the creatine system the electrode system of which comprises only the enzymes creatinase and sarcosine oxidase, since in biological samples creatinine and creatine usually coexist and, according to the system, the creatinine system is only capable of recognizing the sum of creatinine and creatine.
Thus, for a calibration of the biosensor, solutions for calibrating the creatinine, system as well as the creatine system are necessary.
The calibration of a biosensor for the determination of creatinine is aggravated further by the fact that creatinine and creatine are at an equilibrium if the solution has room temperature and a neutral pH, i.e., neither of the two dissolved analytes can, on its own, be maintained at a stable concentration, which, however, would be absolutely necessary for calibrating the electrode systems.
Therefore, the user of the biosensor is forced to freshly prepare the calibration solution(s) before measuring takes place by weighing creatinine and/or creatine into a vessel and dissolving the weighed portion in a certain amount of buffer liquid. It is indeed true that, at room temperature, said solution(s) is (are) sufficiently stable for a short period of time, in general for about a week, since the conversion of creatinine to creatine or vice versa proceeds very slowly, however, the preparation procedure must be repeated after that time period.
Said method is disadvantageous in that the continuous, at least weekly, fresh preparation or the calibration solutions prevents the calibration from being carried out at any time in a quick, simple and topically flexible manner. Furthermore, repeated weighing-in and dissolving with the purpose of securing a sufficiently high accuracy require a larger consumption of creatinine and/or creatine and of buffer liquid.